Recombinant polypeptide compositions are increasingly being used in a wide variety treatments or therapies across the health-related fields. Recombinant polypeptides are being used in diagnostic procedures, as tools in preventative medicine, and to directly save lives through administrative therapies. In addition, recombinant polypeptides are found in a wide array of both health and cosmetic products, used to increase the quality of life. Complex polypeptide products are also routinely used in research laboratories both as end-products of analyses themselves and as agents in assays for the study or preparation of other molecules. Such uses often lead to the discovery of the causes of disease and an understanding of the underlying disease mechanism(s), furthering development toward diagnoses and/or viable treatments. Recombinant polypeptide products also play a vital role in a variety of industrial settings, in areas ranging from farming to the processing of food materials and the raising of livestock to the catalytic degradation of both natural and synthetic by-products and waste materials.
The production of polypeptides for preclinical and clinical evaluation often requires multigrain quantities [Kelley, Bio/Technology 14: 28-31 (1996)]. Industrial applications using such polypeptides generally require even greater quantities and the costs of production are often prohibitive. While there are a variety of ways to chemically synthesize simple polypeptides when the amino acid sequence is known, this method of production has problems with respect to larger polypeptides, e.g., uncertain deviations from native conformational folding, an absence of intracellular post-translational modification, and reduced or limited bioactivity. For these and other reasons, recombinant DNA technology is the most common production method of choice and offers the greatest potential for large-scale production at high efficiency and reasonable cost. Accordingly, the production of useful quantities of these important polypeptides is typically generated through standard recombinant DNA technology, especially where the polypeptide of interest is ultimately modified or where it only occurs naturally in very small amounts.
Current recombinant DNA techniques used for expressing polypeptides can exhibit numerous limitations, including, for example, significant production costs for materials and reagents and low product yield. In addition, production can be time consuming and can require substantial monitoring with limited control. These, as well as other problems and limitations involved in production of recombinant polypeptides, along with inefficiencies in current methods for producing recombinant polypeptides, ultimately can result in a significant toll in costs, resources, health and life itself. Thus, given the fundamental role and countless uses for recombinant polypeptides and the limitations current methods for recombinant polypeptide production, it is of primary importance to employ a method of recombinant gene expression that maximizes protein production and, ultimately, saves time, money and other resources. Accordingly, there exists a need for methods that can increase, maximize and/or optimize the production of recombinant polypeptides.
Several factors can influence recombinant expression in mammalian cells, including promoter strength, the context of the translation initiation region, the efficiency of the 3′ untranslated region to polyadenylate and terminate transcription, the insertion site of the randomly integrated recombinant gene in the host chromosome, and the number of integrated copies of the gene that is being expressed. Of these factors, the choice of promoter and 3′ untranslated regions can significantly impact expression levels. Viral promoters are often used because they are thought to promote high expression levels. The optimal translation initiation sequence, ACCATGG, also known as the Kozak box, can promote more efficient polypeptide synthesis and, therefore, higher expression levels.
One strategy employed for increasing expression of polypeptides uses expression vectors containing multiple integrated copies of a desired gene. Improvements in recombinant polypeptide expression in mammalian cells can be achieved in this manner by effectively increasing the gene dosage in a transfected host cell. Increases in gene copy number are most commonly achieved by gene amplification using cell lines deficient in an enzyme such as dihydrofolate reductase (DHFR) or glutamine synthetase (GS) in conjunction with expression vectors containing genes encoding these enzymes and agents such as methotrexate (MTX), which inhibits DHFR, and methionine sulfoxamine (MSX), which inhibits GS. Using expression vectors containing the recombinant gene under control of a strong promoter and genes encoding DHFR or GS, DHFR+ and GS+ transfectants, respectively, are first obtained and gene amplification is then achieved by growing the transfectants in progressively increasing concentrations of MTX or MSX.
While gene amplification can result in higher levels of expression, it has several drawbacks. First, cell lines that have mutations in the genes encoding the selective enzymes are generally used for gene amplification. In the case of DHFR, both chromosomal copies need to be mutated and, consequently, these cell lines can be less robust than wildtype cells. This can ultimately lead to cells which secrete lower net amounts of the protein of interest as compared to more robust cells that thrive and are stable. In the case of GS, the lymphoid cell line NSO is naturally GS−, but CHO-K1, another commonly-used cell line, is GS+ and requires selection directly for MSX-resistant transfectants. A second problem with current methods of gene amplification is that the amplification can result in cell lines that are unstable in the absence of selective pressure, thus requiring the maintenance of selective pressure. Finally, current methods for gene amplification can be exceedingly time-consuming and can require up to several months to complete with a proportional allocation of both resources and costs. Another strategy for increasing expression of polypeptides uses sequential transfections with expression vectors, each containing single copies of a desired gene but with different selective marker genes such as gpt, neo and his. Gene copy number for the first transfection is one, from the second transfection, two and so on. While this approach can achieve higher levels of expression, it has drawbacks in that the number of gene copies increases only modestly even after three sequential transfections.
Therefore, in view of all these problems, novel ways to increase gene expression through increased gene copy number by methods that do not depend on the use of mutant cell lines and current methods for gene amplification would be highly desirable.
For all the foregoing reasons, there is a need in the art for improved methods of expressing polypeptides of interest at increased net concentrations from stable cell lines, while maximizing efficiency and minimizing expenditure of time, resources, and costs in production. Furthermore, there is a need for expression vectors and stable cell lines harboring and/or integrating into their chromosomes such vectors that are capable of efficiently producing secreting high concentrations of valuable and useful polypeptide products.